For several years our laboratory has focused on the role of orphan nuclear receptors in endocrine development and physiology with an emphasis on members of subfamily V (NR5) that includes steroidogenic factor 1 (SF-1), liver receptor homolog 1 (LRH-1) and the Drosophila Ftz-F1. Broadly speaking, these receptors participate in developmental events as well as in adult functions, including steroid and bile acid homeostasis. However, it has remained unclear if NR5 receptors are ligand-dependent. In a close collaborative effort with Dr. Robert Fletterick's lab at UCSF, we recently obtained the high-resolution crystal structure of the ligand binding domain (LBD) of LRH-1 to 2.4 A resolution, and find that LRH-1 adopts an active conformation in the absence of ligand. Now, we have obtained the crystal structure of mSF-1 LBD to 1.2 A resolution bound by SHP-1 peptide. Comparison of the SF-1 and LRH-1 structures reveals overall similarities, as well as some striking differences. By contrast to the empty pocket observed for mLRH-1, the mSF-1 pocket is occupied by phospholipid phosphotidyl glycerol (PG). Moreover, the region in mSF-1 spanning helices H2 and H3 is much more disordered and flexible than that found in mLRH-1. Here, we propose to extend our structural analyses of NR5 receptors. In our first aim we will assess how differences in helices H2 and H3 affect receptor function, and obtain the crystal structure of coregulatory peptides bound to SF-1/LRH-1, as well as Drosophila, Ftz-F1. Our second aim will address the potential relevance of phospholipids as bona fide ligands for NR5 receptors using biochemical and physiological assays. Finally, in our third aim, we will focus on defining structural changes that occur following posttranslational modification of these receptors, including sumoylation and phosphorylation. We believe that the structural and biochemical studies proposed here will complement greatly the emerging biological framework for this important and biomedical relevant subfamily of nuclear receptors.